Genetic association between asthma and alopecia areata: A two‐sample Mendelian randomization study

Abstract Background Many patients with asthma experience alopecia areata (AA) in their lives. However, it is unclear whether asthma causes or results from AA. Our objective was to investigate the genetic causal relationship between asthma and AA. Methods Two‐sample Mendelian randomization (MR) was used to assess the causal relationship between asthma and AA based on the largest publicly available genome‐wide association study summary statistics. Androgenetic alopecia (AGA) and cicatricial alopecia (CA) were chosen as the control groups for AA. The main estimates were obtained using inverse variance weighting meta‐analysis (IVW), Mendelian randomization‐Egger (MR‐Egger), maximum likelihood estimation, and the weighted median. Sensitivity analyses were conducted using Cochran's Q test, MR‐Egger, and leave‐one‐out methods. Lastly, we conducted a reverse MR analysis to evaluate the possibility of reverse causation. Results Genetically, asthma is associated with an increased risk of AA, while the association between genetically predicted AGA or CA and asthma was negative. The risk of AA increased by 1.86 times in patients with asthma under the IVW method (OR = 1.86, 95% CI = 1.31–2.629, p < 0.001). The reverse MR analysis did not find evidence supporting reverse causality from three phenotypes of alopecia to asthma. Sensitivity analyses yielded consistent causal estimates. Conclusion This study suggests that asthma is causally associated with AA. The findings deepen our understanding of the role of asthma in the pathology of AA, which emphasizes the potential for opening a new vista for the prevention and diagnosis of AA.


INTRODUCTION
Alopecia areata (AA) is an autoimmune disease characterized by transient, non-scarring hair loss and hair follicle preservation.Clinical severity ranges from small patches of hair loss to diffuse or total hair loss.AA affects almost 2% of the general population. 1 The exact mechanisms of alopecia areata are still under investigation.Complex genetic susceptibility, 2 oxidative stress injury, 3 environmental factors, 4 lifestyle, 5 and advanced age contribute cooperatively to the occurrence and severity of AA, 6 among which autoimmune and allergic diseases may play an important role in its pathological changes. 7thma is one of the most common chronic non-communicable diseases in the world, characterized by variable airflow limitation and respiratory symptoms. 8Autoimmunity and allergy play a major role in its development.Studies have reported an association between AA and asthma. 9Asthma was one of the most common comorbidities in patients with AA reported in a US retrospective cross-sectional study. 10However, the relationship between asthma and AA has not been well studied.Therefore, we used Mendelian randomization (MR) to determine whether there is a causal relationship between asthma and AA.
MR is an instrumental variable (IV) analysis method that uses single nucleotide polymorphisms (SNPs) from genome-wide association study (GWAS) as instruments, which are representative of the exposure characteristics, to detect causal relationships between complex characteristics, provided the necessary assumptions are met. 11IVs are not correlated with random error (the presence of variation such as age, sex, and weight, which must be adjusted for in observational studies) because SNP assignment follows the Mendelian inheritance rule, suggesting random assignment during the formation of a fertilized ovum. 12Therefore, the basic principle of MR research based on the SNP acting as an IV is comparable to that of randomized controlled trials.This strategy is relatively convenient, cost-effective, and less likely to be confounded by covariates. 13 this study, we used available summary statistics from open-access GWAS databases to perform a two-sample MR analysis and evaluate the causality between asthma and AA.In order to investigate the relationship between asthma and alopecia areata in more detail and to further explore the mechanism of association between asthma and alopecia areata, we selected androgenetic alopecia (AGA) and cicatricial alopecia (CA) as control groups.The results of the current study may partly explain the underlying mechanism of AA and provide support for the future development of prevention and intervention strategies.

Study design and data source
We performed a Mendelian randomization analysis to investigate the causal effects of asthma on AA and other types of alopecia.
The IVs used for the MR study should meet the following three assumptions 11 : 1.The trait is strongly and robustly associated with the exposure of interest; 2. There are no confounders in the association between IVs and outcomes; 3. IVs do not affect the outcome except through their effect on the exposure (Figure 1).
The MR approach uses genetic variants, which are randomly assigned at meiosis and therefore independent of potential confounders that bias observational studies, as proxies for a risk factor in an instrumental variable analysis.1.
To satisfy assumption 1, we selected SNPs strongly associated with asthma based on the genome-wide significance threshold of p = 5e −8 .
Linkage disequilibrium clustering with a threshold of r 2 < 0.001 and a window size of 10,000 kb was used to determine whether the SNPs were genetically linked or not, with an F-statistic > 10 used as a threshold for sufficient power, representing a 10% relative bias toward the null in the two-sample MR setting. 15In this study, we also used the I 2 statistic (the threshold was 90%) to assess the suitability of pooled data for two-sample Mendelian randomization analyses. 16A higher I 2 statistic means less bias due to weak instruments.We also removed SNPs associated with potential confounders of the outcome of interest according to the PhenoScanner V2 database. 17

Statistical analyses
Main estimates were obtained using inverse variance weighting (IVW), weighted median, Mendelian randomization Egger (MR-Egger) 18, and Maximum Likelihood. 19Random-effects IVW was used when there was heterogeneity between the SNPs included in each analysis.Otherwise, a fixed effects IVW model was used.This method provided the highest statistical power and also accounted for balanced pleiotropy when the above MR assumptions were met.The weighted median used most SNPs (majority of genetic variants) to determine the presence or absence of causality.MR-Egger regression was mainly used to assess horizontal pleiotropy and provide the I 2 statistic to assess weak instrumental variable bias.Maximum likelihood was used to assess bias due to sample overlap. 20 Sensitivity analyses were performed to assess heterogeneity, pleiotropy, and outlier SNPs. 21The Cochran Q statistic was used to test for heterogeneity.A p value < 0.05 was considered to indicate statistically significant heterogeneity.The presence of horizontal and directional pleiotropy violated assumption 3. The intercept in the MR-Egger analysis was used to assess directional pleiotropy.If the intercept was not significantly different from zero (p > 0.05), the pleiotropy effect could be ignored. 18The leave-one-out test was used to assess the influence of outliers.Scatterplots, forest plots, and funnel plots were created to further demonstrate the sensitivity of the results.
Finally, a reverse MR analysis was performed to evaluate the possibility of reverse causation.

F I G U R E 2
Main estimates of Mendelian randomization between asthma and types of alopecia.nSNP, number of single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval; AA, alopecia areata; AGA, androgenic alopecia; CA, cicatricial alopecia; IVs, instrumental variables; IVW, inverse variance weighting; ML, maximum likelihood; WM, weighted median.
All statistical analyses were performed using the "two-sample MR" and "Mendelian randomization" packages in R software (version 4.3.2).

Genetically asthma is associated with increased risk of AA, but not with AGA and CA
During instrumental variable clumping, no SNPs were associated with confounding factors and outcomes, 1 SNP (rs35320232) was removed for incompatible alleles and 1 SNP (rs13099273) was removed for being palindromic with intermediate allele frequencies.Finally, 78 asthma-related SNPs that met the three main assumptions of the MR study were retained for further analysis (Table S1).The F-statistic value of all SNPs was above 10.I 2 statistic was 90.4% which meant low weak instruments bias in further analysis.We used the two-sample MR method to evaluate the potential causal relationship between asthma and AA.The MR analyses suggested a causal relationship between asthma and AA.The risk of AA increased in patients with asthma under the fixed effects IVW method (OR = 1.859, 95% CI = 1.315−2.629,p < 0.001).The causal association was confirmed using both maximum likelihood estimation (OR = 1.817, 95% CI = 1.288−2.565,p = 0.001) and the weighted median method (OR = 2.365, 95% CI = 1.383−4.045,p = 0.002) (Figure 2).When we set the rate of sample overlap as 60%, the causal relationship still existed (OR psi0.6 = 1.212, 95% CI psi0.6 = 1.036−2.195,p psi0.6 = 0.033).
However, we assessed the relationship between asthma and AGA, as well as asthma and CA, using the same MR methods.The results of the fixed effects IVW, the MR-Egger, the weighted median, and the maximum likelihood estimation suggested that there was no clear causal relationship between genetically predicted asthma and the risk of AGA and CA, with all p values greater than 0.05 (Figure 2).The scatter plots demonstrate the impact of the 76 SNPs on asthma and AA, as well as on AGA and CA (Figure 3A-C).The funnel plots illustrate the distribution of the effect of a single SNP (Figure S1A-C), while the forest plots display the effect of an individual SNP on the estimation of the outcomes (Figure S1D-F).

Sensitivity analysis
The analysis did not reveal any significant heterogeneity among the asthma-related SNPs (Cochran's Q statistic was 80.441, p = 0.285, MR-Egger method).Furthermore, the MR-Egger intercept analysis found no evidence of directional pleiotropy (egger intercept = −0.019,p = 0.537) in MR analysis between asthma and AA, suggesting that F I G U R E 3 Scatter plots about the causal effect of asthma on types of alopecia.(A) Scatter plots about the causal effect of asthma on alopecia areata.(B) Scatter plots about the causal effect of asthma on androgenic alopecia.(C) Scatter plots about the causal effect of asthma on cicatricial alopecia.
potential confounding factors through other pathways were unlikely to affect the exposure-outcome relationship (Table 2).
The leave-one-out analysis indicated that no single SNP had a significant effect on the results, suggesting the robustness and reliability of the MR studies (Figure S1G-I).The reverse MR analysis did not find any evidence supporting reverse causality from three phenotypes of alopecia to asthma (Table S2, Figures S2 and S3).

DISCUSSION
Alopecia, or hair loss, is a disease that affects a significant number of people worldwide and is associated with psychological disorders. 22ere are two main types of alopecia: CA and non-cicatricial alopecia.
The most common types of non-cicatricial alopecia are AA and AGA.
The causes of alopecia are complex and involve multiple factors.
Autoimmune and inflammatory diseases, including asthma, may contribute to alopecia.To our knowledge, this is the first MR study to investigate the potential causal relationship between asthma and alopecia.Our study showed that genetically predicted asthma increases the risk of AA later in life, but not of AGA and CA.The results were supported by consistent magnitude and direction of robust methods.
AGA, also known as male-pattern alopecia, is the most common type of alopecia. 23It is characterized by progressive terminal hair loss after puberty, and its incidence and prevalence depend on race and age.At least 80% of men and 50% of women are affected by the age of 70 years.The progression of AGA is mainly contributed by genetics and sensitivity to androgens, 24 with testosterone being a major androgen that is converted to dihydrotestosterone (DHT) by 5α-reductase.DHT binds to androgen receptors in hair follicles, causing them to shrink and shortening their anagen cycle, ultimately Abbreviations: AA, alopecia areata; CA, cicatricial alopecia; AGA, androgenic alopecia; IVW, inverse variance weighting.
resulting in hair loss. 25Some metabolic disorders may also have an influence on AGA by elevating androgens or other metabolites. 26 is important to note that the pathogenesis of AGA differs from that of AA, which is primarily considered an autoimmune disease.
This may explain why no association was found between asthma and AGA.
Scarring alopecia, also known as CA, is divided into primary and secondary forms.It is characterized by irreversible inflammation and damage to the hair follicles, resulting in scarring and hair loss.Primary CA is primarily caused by direct inflammatory attacks on hair follicles by various inflammatory infiltrates, such as lymphocytes, neutrophils, and mixed cells.This can occur in diseases such as chronic cutaneous lupus erythematosus, lichen planopilaris, folliculitis decalvans, and folliculitis (acne) necrotica. 27Secondary CA results from the random destruction of hair follicles due to inflammatory processes or mechanical damage, such as trauma, burns, radiation, infections, or tumors. 28ile autoimmune and inflammatory diseases can cause CA, the pathogenesis of CA differs from that of non-cicatricial alopecia.Our results demonstrate that asthma does not increase the risk of CA, indicating that asthma may not cause CA.
Previous observational studies have shown that AA is associated with autoimmune and inflammatory diseases, including asthma.A Danish nationwide register-based cohort study found that asthma occurred more frequently in patients with AA compared to controls. 9milarly, a population-based study including 51,561 cases with AA and 51,410 matched controls found that the prevalence of asthma was significantly higher in patients with AA than in the control group. 29rthermore, a recent retrospective cohort study based on US claims data demonstrated that the incidence of asthma was higher in individuals with African ancestry compared to those without (12.8% vs. 8.8%, respectively). 7Although observational studies may be subject to recall bias, our study was based on a large GWAS dataset and a restricted population to minimize bias.Our findings are consistent with previous research, indicating that asthma is a risk factor for African ancestry.No observational studies have shown an association between asthma and AGA or CA.
The association between asthma and AA is not well understood, but there are some mechanisms that can explain the connection.An explanation is the presence of genetic variants in immune-related genes that have been linked to both conditions.Previous studies have shown that the cytotoxic T-lymphocyte protein 4 (CTLA-4) gene polymorphism is associated with asthma. 30,31CTLA-4 is expressed on activated T cells and is involved in T cell activation and differentiation, which plays an important role in autoimmune diseases.Studies have also demonstrated a genetic variant association of CTLA-4 with AA. 32 Other genetic variants, such as HLA-DRB1 and PTPN22, are associated with an increased susceptibility to asthma and AA. 33This supports the association between asthma and AA in the present study.Additionally, both asthma and AA are considered autoimmune diseases, suggesting that they may share similar pathogenesis and immune pathways.Th2 cells play an essential role in the pathogenesis of asthma.They produce IL-4, IL-5, and IL-13 under adaptive immune response, leading to airway obstruction and hyperresponsiveness. 34 These cytokines are also involved in AA pathogenesis, as levels of IL-4, IL-5, and IL-13 have been found to be elevated in the lesions or serum/blood of patients with AA. 35 Several clinical trials have shown that Dupilumab, a monoclonal antibody that targets IL-4 and IL-13, is effective in treating asthma. 36,37Recent studies have also demonstrated its effectiveness in treating AA and indicated the involvement of the Th2 pathway in AA pathogenesis. 38,39In addition, Th1 and Th17 cells also play an important role in asthma, particularly in those with lower Th2 immune response, mainly producing IFN-γ, IL-17, and IL-22.Patients with AA also exhibit elevated levels of Th1 and Th17-related cytokines, which are essentially involved in the pathogenesis of AA. 40,41 The changes in immune system may cause a breakdown of immune privilege of hair follicles, which have been thought to be a crucial driver of AA. 1,42 These mechanisms may form the foundation of causal association between asthma and AA.
Our study has several strengths.First, the MR concept is less susceptible to unobserved confounding and reverse causality, which are issues in observational studies.Second, we conducted outlier assessment and a wide range of sensitivity analyses that accounted for different patterns of pleiotropy, which increased the robustness of the results and strengthened the evidence of our findings.There are still shortcomings in our research.First, MR studies have some disadvantages compared to randomized clinical trials, such as lack of stability of subjects or weak controllability of studies.Second, the number of cases from summary data used in this study was lower than anticipation, which is attributed to low incidence of alopecia areata.Additionally, due to the lack of suitable summary-level data, sex-specific and ethnicstratified analyses were not possible.Future high-quality randomized controlled studies based on large sample sizes are still needed to further verify this association.

CONCLUSION
This study suggests that asthma has a positive effect on AA, while it has no association with the risk of AGA and CA.Furtherly exploration into the role of asthma in AA will promote an understanding of its pathogenesis.The findings enhance our comprehension of the relationship between asthma and the pathology of AA, highlighting the possibility of new avenues for the prevention and diagnosis of AA.

F I G U R E 1
Assumptions and research design for Mendelian randomization analysis.(A) In order to verify the existence of causal correlation, it is necessary to meet the conditions as follows: (1) The trait is strongly and robustly associated with the exposure of interest; (2) There are no confounders of the association between IVs and outcome; (3) IVs does not affect the outcome except through its effect on the exposure.(B) Flowchart of Mendelian randomization analysis and sensitivity analyses.SNPs, single nucleotide polymorphisms; GWAS, genome-wide association study; AA, Alopecia areata; AGA, Androgenic alopecia; CA, Cicatricial alopecia; IVs, instrumental variables; IVW, inverse variance weighting.
Sensitive analysis for heterogeneity, pleiotropy and outlier SNPs.
TA B L E 2